Two novel fusion transcripts for early detection of non-small-cell lung cancer (NSCLC)

Abstract

The present invention provides a new gene fusion of TPTE2 and MRPS31P2 which is characteristic for Non-small-cell lung cancer cells and can be used for specific detection of Non-small-cell lung tumors.

Description

[0001] Two novel fusion transcripts for early detection of Non-small-cell lung cancer (NSCLC)

[0002] Background of the Invention

[0003] Lung cancer remains the leading cause of cancer deaths worldwide [1]. It is classified into Non-small-cell lung cancer (ca. 85% of cases) and small-cell lung cancer (ca 15% of cases). Non-small cell lung cancer (NSCLC) grows slowly compared to small cell lung cancer — but it often spreads to other parts of the body by the time it is diagnosed. Therefore, early detection and treatment are essential.

[0004] NSCLC can be divided into three main kinds [2,3], Adenocarcinomas (40% of cases) are most common, start to grow in the gland cells that secrete mucus and are usually found in the outer parts of the lungs. Squamous cell carcinomas (25-30% of cases) are often linked to smoking, start to grow in squamous cells (flat cells lining the airways) and are usually found in the central parts or in the bronchial tubes. Large cell carcinomas (5-10% of cases) are rare, tend to grow rapidly and can be found anywhere in the lungs.

[0005] NSCLC is often not detected until the tumor has developed into an advanced state causing symptoms like cough, hemoptysis, dyspepsia or achy chest [4], The five- year survival of lung cancer is 18%, representing one of the worst of any cancer [1], Early detection could greatly improve the survival rate of patients. Thus, novel diagnostic markers for early detection of NSCLC are highly needed.

[0006] Short Descrintion of the Invention

[0007] Based on RNA-Sequencing data from NSCLC biopsies, the present invention provides new information on gene fusions associated with NSCLC which can be used for early cancer detection.

[0008] The first fusion is a fusion between TPTE2 and MRPS31P2 which are fused between nucleotide 13: 19475573 and nucleotide 13: 19570288. The second fusion is a fusion between SVEP1 and MYLK which are fused between nucleotide 9: 110417363 and nucleotide 3: 123831670.

[0009] The present invention provides methods for detecting said gene fusions by means of PCR, sequencing, target enriched sequencing and in situ hybridization. Detecting the presence of said gene fusion in liquid biopsies or tissue, in particular lung tissue may be indicative for a cancer, preferably for a lung cancer and most preferably a non small cell lung cancer (NSCLC).

[0010] Thus, in a first aspect, the present invention provides an isolated nucleic acid comprising a gene fusion consisting of TPTE2 and MRPS31P2, or a gene fusion consisting of SVEP1 and MYLK. Usually, nucleotide 13: 19475573 is fused to nucleotide 13: 19570288. Alternatively the isolated nucleic acid comprises a gene fusion consisting of SVEP1 and MYLK. Then, nucleotide 9: 110417363 is usually fused to nucleotide 3: 123831670.

[0011] In a another aspect, the present invention provides a method for detecting a gene fusion consisting of TPTE2 and MRPS31P2, comprising the steps of (i) providing a nucleic acid sample which comprises human nucleic acid, (ii) adding a first amplification primer which is identical to a part of Seq. Id. No: 1, which is derived from TPTE2, (iii) adding a second amplification primer which specifically hybridizes to the complement of a part of of Seq. Id. No: 1, which is derived from MRPS3 1P2, (iv)performing a nucleic acid amplification reaction by means of using said two amplification Primers, and (v) detecting whether amplification has occurred. One of said primers may be identical to Seq. ID. No: 3 and the other of said primers is identical to Seq. ID. No: 4.

[0012] Alternatively, the present invention provides a method for detecting a gene fusion consisting of SVEP1 and MYLK, comprising the steps of (i) providing a nucleic acid sample which comprises human nucleic acid, (ii) adding a first amplification primer which is identical to a part of Seq. Id. No: 2, which is derived from SVEP1, (iii) adding a second amplification primer which specifically hybridizes to the complement ofa part of Seq. Id. No: 2, which is derived from MYLK, (iv)performing a nucleic acid amplification reaction by means of using said two amplification Primers, and (v) detecting whether amplification has occurred. One of said primers may be identical to Seq. ID. No: 6 and the other of said primers is identical to Seq. ID. No: 7.

[0013] In a further aspect, the present invention provides a method for detecting a gene fusion consisting of TPTE2 and MRPS31P2 or SVEP1 and MYLK, comprising the steps of (i) providing a nucleic acid sample which comprises human nucleic acid, (ii) sequencing said nucleic acid, (iii) determining whether said sample comprises a gene fusion consisting of TPTE2 and MRPS31P2 or SVEP1 and MYLK. In a still further aspect, the present invention provides a method for detecting a gene fusion consisting of TPTE2 and MRPS31P2, comprising the steps of (i) providing a nucleic acid sample which comprises human nucleic acid, (ii) hybridizing said nucleic acid with at least 2 capture probes wherein one capture probe specifically hybridizes to TPTE2 and the other one specifically hybridizes to MRPS31P2, (iii) enriching for nucleic acid hybridized to said capture probe sequencing said hybrids, and determining whether said sample comprises a gene fusion consisting of TPTE2 and MRPS31P2.

[0014] Similarly, a method for detecting a gene fusion consisting of SVEP1 and MYLK is provided, comprising the steps of (i) providing a nucleic acid sample which comprises human nucleic acid, (ii) hybridizing said nucleic acid with at least 2 capture probes wherein one capture probe specifically hybridizes to SVEP1 and the other one specifically hybridizes to MYLK, (iii) enriching for nucleic acid hybridized to said capture probe sequencing said hybrids, and determining whether said sample comprises a gene fusion consisting of SVEP1 and MYLK.

[0015] Alternatively, a method for detecting a gene fusion consisting of TPTE2 and MRPS31P2 or SVEP1 and MYLK comprises the steps of (i) providing a nucleic acid sample which comprises human nucleic acid, (ii) hybridizing said nucleic acid with at least one capture probe that specifically hybridizes to a nucleic acid which comprises a gene fusion between TPTE2 and MRPS31P2 or SVEP1 and MYLK , (iii) enriching for hybrids consisting of said capture probe and nucleic acid hybridized to said capture probe (iv) sequencing said hybrids, and (v) determining whether said sample comprises a gene fusion consisting of gene TPTE2 and MRPS31P2, SVEP1 and MYLK.

[0016] The nucleic acids that are analyzed according to the methods disclosed above may be RNA or cDNA. The nucleic acid may originate from a sample is derived from a biopsy, which may be a liquid biopsy such as blood, serum or plasma. Alternatively, the said sample is derived from lung tissue.

[0017] The present invention also provides a method for detecting a gene fusion consisting of TPTE2 and MRPS31P2 and / or SVEP1 and MYLK , comprising the steps of (i) providing a tissue sample derived from lung tissue, (ii) executing an in situ hybridization with a probe that specifically hybridizes to a nucleic acid which comprises a gene fusion between TPTE2 and MRPS31P2 and / or SVEP1 and MYLK, and (iii) determining whether said sample comprises a gene fusion consisting of TPTE2 and MRPS31P2 and / or SVEP1 and MYLK

[0018] The detection of the presence of one of said gene fusions will be indicative for a cancer, and in particular a lung cancer such as a non small cell lung cancer (NSCLC).

[0019] Furthermore, it is also within the scope of the present invention, if the TPTE2 and MRPS31P2 fusion as well as the fusion between SVEP1 and MYLK are detected simultaneously by any of the methods disclosed above.

[0020] Detailed Descrintion of the Invention

[0021] In order to identify nucleic acids which are overexpressed in NSCLC (non small cell lung cancer), a whole transcriptome analysis on early stage NSCLC cells was performed. Total RNA sequencing data from NSCLC biopsies were compared with data from adjacent normal tissue to identify RNAs that are tumor specific.

[0022] Samples from patients with NSCLC were obtained from two cohorts. Both cohorts contain tumor tissue and adjacent normal tissue. The distance between the origins of the biopsies was approximately 1 cm. A first cohort contained samples from 71 patients from patients with stage 1 NSCLC (Indivumed cohort). The patients can be further discriminated into 43 patients with Adenocarcinoma, 25 patients with Squamous cell carcinoma and 3 patients with Large cell carcinoma, which roughly represents the total abundance of each subtype. The second cohort contained samples from 199 patients with stage 1, 2 and 3 NSCLC (Heidelberg cohort). The patients can be further discriminated into 129 Adenocarcinoma, 34 Squamous cell cancer and 36 neuroendocrine tumors.

[0023] RNA from fresh-frozen samples was isolated using the RNeasy Mini Kit (Qiagen). Library preparation was done using lOOng of isolated RNA per sample with the KAPA RNA HyperPrep with RiboErase (HMR) (Roche). Libraries were sequenced on a NovaSeq6000 with paired-end reads of length lOObp to a depth of 100-200M per sample. Demultiplexing of the sequencing reads was performed with Illumina bcl2fastq (2.20). Deep total RNA-seq was applied on each of the samples with a sequencing depth of 100 000 000 -200 000 000 reads.

[0024] Computational analysis of the sequence data obtained was focused on the identification of RNAs that are specific for tumor samples and not expressed in the rest of the body. In particular, computational analysis was performed to identify potential fusion transcripts potentially originating from NSCLC specific DNA gene fusions. More precisely, Two tools (STAR-fusion [9] and Arriba

[0010] ) were selected for the analysis based on an assessment in Haas et al

[0011] ,

[0025] STAR-Fusion is a component of the Trinity Cancer Transcriptome Analysis Toolkit (CTAT). STAR-Fusion uses the STAR aligner to identify candidate fusion transcripts supported by Illumina reads. STAR-Fusion further processes the output generated by the STAR aligner to map junction reads and spanning reads to a reference annotation set.

[0026] Arriba is a command-line tool for the detection of gene fusions from RNA-Seq data. It is based on the ultrafast STAR aligner. Arriba's workflow produces fully reusable alignments, which can serve as input to other common analyses, such as quantification of gene expression. Arriba does not require to reduce the STAR parameter — alignlntronMax to detect fusions arising from focal deletions. Reducing this parameter impairs mapping of reads to genes with long introns and may affect expression quantification, hence.

[0027] Raw sequencing reads were processed using the rnafusion pipeline from nf-core (https: / / nf-co.re / rnafusion / 2.3.4), a bioinformatics best-practice analysis pipeline for RNA sequencing analysis pipeline with curated list of tools for detecting and visualizing fusion genes.

[0028] The parameters used for both tools are as follows: a. STAR-fusion nextflow run nf-core / mafusion —input

[0029] / tmbd / barbara / LC / Indivumed / samplesheet.csv —starfusion — outdir Indivumed LC rnafusion — skip vis — genomes base / tmbd / barbara / STARFusion / rnafusion references -profile singularity b. Arriba nextflow run nf-core / mafusion —input

[0030] / tmbd / barbara / LC / Indivumed / samplesheet.csv — arriba —outdir Indivumed LC rnafusion — skip vis — genomes base / tmbd / barbara / STARFusion / rnafusion references -profile singularity Several fusion transcripts were identified using both algorithms. Two of them were identified in 18 and 16 tumor samples, respectively, compared to only one in normal adjacent tissue sample (for fusion TPTE2 — MRPS31P2) in the first cohort. The same fusion were identified in the second cohort in 77 and 18 tumor samples, respectively, compared to only 8 in normal adjacent tissue (for fusion TPTE2 — MRPS31P2).

[0031] Using these detection algorithms, two different fusions were identified. The first fusion is a fusion between sequences encoded by TPTE2 and MRPS31P2 which are fused between nucleotide 13: 19475573 and nucleotide 13: 19570288. The second fusion is a fusion between sequences encoded by SVEP1 and MYLK which are fused between nucleotide 9: 110417363 and nucleotide 3: 123831670.

[0032] The structure of the first fusion (TPTE2 — MRPS31P2) is shown in Fig. 1 The fusion RNA was identified in 18 tumor samples and 1 normal adjacent tissue with Arriba and in 9 tumor samples with STAR-fusion in the first cohort. In the second cohort, it was identified in 77 tumor samples and 8 normal adjacent tissue samples. The nucleic acid sequence of the first five exons of the protein coding gene TPTE2 (transcript ENST00000382978) fuses to the upstream non-coding RNA MRPS31P2.

[0033] The structure of the second fusion (SVEP1 — MYLK ) is shown in Figure 2. The fusion transcript was identified in 16 tumor samples, but no normal adjacent tissue with Arriba in the first cohort. In the second cohort, it was identified in 15 tumor samples and no normal adjacent tissue samples. The first 36 exons and some part of the downstream intron of the nucleic acid sequence encoding protein SVEP1 on chromosome 9 fuse to the 3rdexon of the nucleic acid encoding protein MYLK on chromosome 3. The exact fusion point is located in the intron after exon 36 of SVEP1. The fusion points overlaps a transposable element. Several fusion transcripts are described already with gene MYLK, however no SVEP1-MYLK fusion has been disclosed so far.

[0034] In order to check, whether these fusions had been identified and described previously, the following sources were checked.

[0035] • COSMIC (Catalogue of Somatic Mutations in Cancer) [5], a database with manually curated known mutation and fusion transcripts

[0036] • Mittelmann database of chromosome aberrations and gene fusions in cancer [6], a database with manually curated data from literature • TumorFusions [7], a data portal that catalogues 20731 gene fusions detected in 9966 well characterized cancer samples and 648 normal specimens from The Cancer Genome Atlas (TCGA)

[0037] • Resource paper from TCGA [8], based on a systematic investigation of fusions in 9,624 tumors across 33 cancer types using multiple fusion calling tools

[0038] None of the references disclosed above recite any fusion between TPTE2 and MRPS3 1P2 or SVEP1 and MYLK. Since the in silico identification disclosed above did not exclude the possibility of a potential sequencing or sequence alignment errors, RT-PCR analysis was performed in order to confirm that the discovered fusions actually originate from real transcripts. Details are disclosed in the Examples section below.

[0039] Due to the exclusive presence of the discovered novel gene fusion in tumor tissue forom NSCLC as compared to surrounding no tumorogenic tissue, methods for detecting said fusion provide an innovative tool for the detection of lung Cancer and in parti clular for the detection of very early stage I NSCLC detection. The nucleic acids to be analyzed may be isolated from a biopsy, which may be a liquid biopsy and may be selected selected from a group consisting of blood, serum or plasma. Alternatively, the sample may be derived from lung tissue.

[0040] One straight forward possibility is detection by means of PCR. This can be done using either genomic DNA, reverse transribed single stranded or double strande cDNA, total cellular RNA or total cellular mRNA (poly-A RNA) as a starting material. Respective methods for isolating and / or preparing such a starting material are known in the art.

[0041] Fusions betweenTPTE2 and MRPS31P2 are detected using a first amplification primer which specifically hybridizes to the part of Seq. Id. No: 1, which is derived from TPTE2, and a second amplifiaction primer adding a second amplification primer which specifically hybridizes to the complement of Seq. Id. No: 1, which is derived from MRPS31P2. Fusion between SVEP1 and MYLK are detected using a first amplification primer which specifically hybridizes to the part of Seq. Id. No: 2, which is derived from TPTE2, and a second amplification primer which specifically hybridizes to the complement of Seq. Id. No: 2, which is derived from MRPS31P2. The PCR performed may be a qPCR reaction which is monitored in real time. In a multiplex approach, both fusions can be detected simutaneously using two differently labeled hybridization probes each specifically hybridizing either to the first amplicon generated by the first pair of amplifiaction primers or to the second amplicon generated by the second pair of amplification primers.

[0042] Another possibility for detecting the novel gene fusions is based on nucleic acid sequencing according to standard methods known in the art. The nucleic acid to be seqeunced represents either total cellular DNA, single stranded or double stranded cDNA, total cellular RNA or total cellular mRNA (poly-A RNA) and may be isolated and / or prepared according th standard methods. If an analysis of the sequencing data reveals the presence and detection of the fusions disclosed above, then this is indicative for the presence of a lung cancer and in particular NSCLC in the patient.

[0043] Total DNA sequencing however, is expensive and can be avoided by target enrichment protocols which capture selective sequences of interest. In the capture method, genomic DNA is fragmented (by physical shearing or enzymatic methods) and prepared for sequencing by adding adapters specific to the sequencing platform used, which typically act as barcodes for later identification. cDNA is usually shorter and thus dous not require a fragmentation treatment. The DNA is then hybridized to single-stranded oligonucleotides (probes or baits that are designed to target specific regions of interest). Typically, these probes are biotinylated and can be recovered using streptavidin-coated magnetic beads, and the process can be used to capture target DNA in the bead complex.

[0044] For the detection of a gene fusion consisting of TPTE2 and MRPS31P2, at least 2 capture probes must be used wherein one capture probe specifically hybridizes to TPTE2 and the other one specifically hybridizes to MRPS31P2. For the detection of SVEP1 and MYLK at least 2 capture probes must be used wherein one capture probe pecifically hybridizes to SVEP1 and the other one specifically hybridizes to MYLK. Alternatively, at least one capture probe is used which specifically hybridizes to a nucleic acid which comprises a gene fusion between either TPTE2 and MRPS31P2 or SVEP1 and MYLK. Furthermore, it is possible to use both types of capture probes simultanouslyithin on reaction set-up.

[0045] In an altemave aproach, the novel gene fusions might also be detected by means of in situ hybridization. The tissue sample to be analyzed must be a lung tissue biopsy . The hybridiation probes to be used specifically hybridize to the sequences upstream and downstream of chromosomal breakpoint of a gene fusion between either TPTE2 and MRPS31P2 or SVEP1 and MYLK. In a multiplex apprach, the probes are used, wherein one hybridizes to the sequences upstream and downstream of chromosomal breakpoint of a gene fusion between TPTE2 and MRPS31P2, abd the other one hybridizes to the sequences upstream and downstream of chromosomal breakpoint of a gene fusion between SVEP1 and MYLK. In the later case, the two probes are preferably labeld with different flourescent tags. Again, it is possible to use both types of capture probes simultanously within on reaction set-up.

[0046] Furthermore, it is also within the scope of the present invention, if the TPTE2 and MRPS31P2 fusion as well as the fusion between SVEP1 and MYLK are detected simultaneously by any of the methods disclosed above.

[0047] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0048] Descrintion of the Figures

[0049] Figure 1 Structural and functional characteristics of TPTE2 — MRPS31P2.

[0050] Upper panel shows the chromosome and cytoband where the breakpoint is located on for each partner gene as well as the predicted fusion structure. The RNA-seq coverage for each exon is shown above the exon structure. The sequence around the fusion break point is given below the fusion gene structure.

[0051] Figure 2 Structural and functional characteristics of SVEP1 — MYLK.

[0052] Upper panel shows the chromosome and cytoband where the breakpoint is located on for each partner gene as well as the predicted fusion structure. The RNA-seq coverage for each exon is shown above the exon structure. The sequence around the fusion break point is given below the fusion gene structure.

[0053] Figure 3 Sequence around the TPTE2 — MRPS31P2 fusion breakpoint (Seq. ID. No: 1)

[0054] The actual breakpoint is indicated in the middle. Locations of the forward and reverse primer and the TaqMan probe are also indicated. PCR amplification results in a 173bp amplicon. Figure 4 Sequence around the SVEP1 — MYLK fusion breakpoint (Seq. ID. No: 2)

[0055] The actual breakpoint is indicated in the middle. Locations of the forward and reverse primer and the TaqMan probe are also indicated. PCR amplification results in a 173bp amplicon.

[0056] Examnle

[0057] Confirmation of fusion transcripts by RT-PCR

[0058] In order to detect the predicted fusion transcripts by means of RT-PCR, primers and probes were designed to create an amplicon which overlaps the fusion point for each fusion transcript. Amplification of the SVEP1 — MYLK fusion results in a 122bp amplicon. Amplification of the TPTE2 — MRPS31P2 fusion results in a 173bp amplicon.

[0059] Exact primers and probes were as follows:

[0060] Table 1: Details for the primers and TaqMan probes used . “TM” Oligos are primers and probe used for amplification of TPTE2 — MRPS31P2. “SM” Oligos are primers and probes used for amplification of SVEP1 — MYLK

[0061] Lung biopsies (tumor sample and normal adjacent tissue per patient) from 4 patients were selected to execute RT-PCR.

[0062] Total RNA from NSCLC biopsies was isolated from 4 different patients. Taken previous sequencing data disclosed above into account, it was expected that the RNA from patients No. 1 and 2 only contained the TPTE2 — MRPS31P2 fusion, but not the SVEP1 — MYLK fusion, whereas No. 3 and 4 contained both TPTE2 fusions. cDNA was prepared by means of a reverse transcription reaction at 60 min 50 °C, 5 min, 85 °C with Transcriptor First Strand cDNA Synthesis Kit (Mat# 04379012001) and oligo(dT) primers. Quantitative PCR was performed on LightCycler® instrument with the LightCycler® 480 Probes Master kit (Mat# 04707494001) using 2 pl of cDNA and 500 nM each primer and 200 nM TaqMan probe and G6PDH as an internal positive control reference gene. The results for the measured average ct values were as follows:

[0063] Table 2: Results of RT-PCR detection of fusion transcripts. The generated ct values invertedly correspond to the amount of RNA detected. The lower the ct value is, the higher is the amount of RNA originally present in the sample. Ct values above 37.5 are considered as negative.

[0064] As can be deduced from the table, the TPTE2 — MRPS31P2 fusion was detectable in tumor tissue from all 4 patients with Ct values of ~30, but it is not detectable in adjacent normal tissue.

[0065] On the other hand, the fusion transcript SVEP1 — MYLK which was predicted to be only expressed in patients 3 and 4 but not patients 1 and 2 was actually indeed only detecable in tumor DNA patients 3 and 4 with Ct values of ~29, bujt neither detecable in normal adjacent tissue nor tumor tissue from patients 1 and 2. Thus, the qPCR results confirm the predictions of regarding the existence of perviously unidentified fusion transcripts in NSCLC cells from small subset of 4 patients and ruled out the possibility of an algorithmic artefact.

[0066] List of References

[0067] [1] Siegel RL, Miller KD, Fuchs HE, Jemal A.

[0068] Cancer statistics, 2022. CA Cancer J Clin. 2022;72:7-33.

[0069] [2] Travis WD, Brambilla E, Nicholson AG, et al.

[0070] The 2015 World Health Organization Classification of Lung Tumors: impact of genetic, clinical and radiologic advances since the 2004 classification. J Thorac Oncol. 2015;10: 1243-60.

[0071] [3] Travis WD, Brambilla E, Burke AP, et al.

[0072] Introduction to the 2015 world health organization classification of tumors of the lung, pleura, thymus, and heart. J Thorac Oncol. 2015;10: 1240-2.

[0073] [4] Kocher F, Hilbe W, Seeber A, et al.

[0074] Longitudinal analysis of 2293 NSCLC patients: a comprehensive study from the TYROL registry. Lung Cancer. 2015;87: 193-200.

[0075] [5] COSMIC: the Catalogue Of Somatic Mutations In Cancer John G Tate, Sally Bamford, Harry C Jubb, Zbyslaw Sondka, David M Beare, Nidhi Bindal, Harry Boutselakis, Charlotte G Cole, Celestino Creatore, Elisabeth Dawson, Peter Fish, Bhavana Harsha, Charlie Hathaway, Steve C Jupe, Chai Yin Kok, Kate Noble, Laura Ponting, Christopher C Ramshaw, Claire E Rye, Helen E Speedy, Ray Stefancsik, Sam L Thompson, Shicai Wang, Sari Ward, Peter J Campbell, Simon A Forbes

[0076] Nucleic Acids Research, Volume 47, Issue DI, 08 January 2019, Pages D941-D947, https: / / doi.org / 10.1093 / nar / gkyl015

[0077] [6] Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (2023). Mitelman F, Johansson B and Mertens F (Eds.), http s : / / mitelmandatab ase.isb-cgc. org

[0078] [7] Hu X, Wang Q, Tang M, Barthel F, Amin S, Yoshihara K, Lang FM, Martinez- Ledesma E, Lee SH, Zheng S, Verhaak RGW. TumorFusions: an integrative resource for cancer-associated transcript fusions. Nucleic Acids Res. 2018 Jan 4;46(D1):D1144-D1149. doi: 10.1093 / nar / gkxl018. PMID: 29099951; PMCID: PMC5753333.

[0079] [8] Gao Q, Liang WW, Foltz SM, Mutharasu G, Jayasinghe RG, Cao S, Liao WW, Reynolds SM, Wyczalkowski MA, Yao L, Yu L, Sun SQ; Fusion Analysis Working Group; Cancer Genome Atlas Research Network; Chen K, Lazar AJ, Fields RC, Wendl MC, Van Tine BA, Vij R, Chen F, Nykter M, Shmulevich I, Ding L. Driver

[0080] Fusions and Their Implications in the Development and Treatment of Human Cancers. Cell Rep. 2018 Apr 3;23(l):227-238.e3. doi: 10.1016 / j.celrep.2018.03.050. PMID: 29617662; PMCID: PMC5916809.

[0081] [9] STAR-Fusion: Fast and Accurate Fusion Transcript Detection from RNA-Seq

[0082] Brian Haas, Alex Dobin, Nicolas Stransky, Bo Li, Xiao Yang, Timothy Tickle, Asma Bankapur, Carrie Ganote, Thomas G. Doak, Nathalie Pochet, Jing Sun, Catherine J. Wu, Thomas R. Gingeras, Aviv Regev bioRxiv 120295; doi: https: / / doi.org / 10.1101 / 120295

[0083]

[0010] Uhrig S. Arriba

[0084] Fast and accurate gene fusion detection from RNA-Seq data 2019. Available from : http s : / / github . com / suhrig / arrib a .

[0085]

[0011] Haas, B.J., Dobin, A., Li, B. et al.

[0086] Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly -based methods. Genome Biol 20, 213 (2019). https: / / doi.org / 10.1186 / sl3059-019-1842-9

Claims

Patent Claims1. An isolated nucleic acid comprising a gene fusion consisting of the TPTE2 and MRPS31P2.

2. An isolated nucleic acid according to claim 1 wherein nucleotide 13: 19475573 is fused to nucleotide 13:19570288.

3. A method for detecting a gene fusion consisting of TPTE2 and MRPS31P2, comprising the steps of providing a nucleic acid sample which comprises human nucleic acid adding a first amplification primer which is identical to a part of Seq. Id. No: 1, which is derived from TPTE2 adding a second amplification primer which specifically hybridizes to a part of Seq. Id. No: 1, which is derived from MRPS31P2 performing a nucleic acid amplification reaction by means of using said two amplification Primers detecting whether amplification has occurred.

4. A method according to claim 3 wherein one of said primers is identical to Seq. ID. No: 3.

5. A method according to claim 3-4 wherein one of said primers is identical to Seq. ID. No: 4.

6. A method for detecting a gene fusion consisting of TPTE2 and MRPS31P2, comprising the steps of providing a nucleic acid sample which comprises human nucleic acid sequencing said nucleic acid determining whether said sample comprises a gene fusion consisting of TPTE2 and MRPS31P2.

7. A method for detecting a gene fusion consisting of TPTE2 and MRPS31P2, comprising the steps ofproviding a nucleic acid sample which comprises human nucleic acid hybridizing said nucleic acid with at least 2 capture probes wherein one capture probe specifically hybridizes to TPTE2 and the other one specifically hybridizes to MRPS31P2 enriching for nucleic acid hybridized to said capture probe sequencing said hybrids, determining whether said sample comprises a gene fusion consisting of TPTE2 and MRPS31P2.

8. A method for detecting a gene fusion consisting of TPTE2 and MRPS31P2 comprising the steps of providing a nucleic acid sample which comprises human nucleic acid hybridizing said nucleic acid with at least one capture probe that specifically hybridizes to a nucleic acid which comprises a gene fusion between TPTE2 and MRPS31P2 enriching for hybrids consisting of said capture probe and nucleic acid hybridized to said capture probe sequencing said hybrids, determining whether said sample comprises a gene fusion consisting of gene TPTE2 and MRPS31P2.

9. A method according to claims 3 - 8 wherein said nucleic acid comprises RNA or cDNA.

10. A method according to claims 3-9, wherein said sample is derived from a biopsy, which is preferably a liquid biopsy and most preferably selected from a group consisting of blood, serum or plasma.

11. A method according to claims 3-9, wherein said sample is derived from lung tissue.

12. A method for detecting a gene fusion consisting of TPTE2 and MRPS31P2, comprising the steps ofproviding a tissue sample derived from lung tissue executing an in situ hybridization with a probe that specifically hybridizes to a nucleic acid which comprises a gene fusion between TPTE2 and MRPS31P2 - determining whether said sample comprises a gene fusion consisting ofTPTE2 and MRPS31P2.

13. Method according to claim 10-12, wherein the presence of said gene fusion is indicative for a cancer, preferably a lung cancer and most preferably a non small cell lung cancer (NSCLC).

14. A method according to claims 3-13, further comprising detecting a gene fusion consisting of SVEP1 and MYLK.

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